Summary

The mammary glands develop initially as buds arising from the ventral
embryonic epidermis. Recent work has shed light on signaling pathways leading
to the patterning and formation of the mammary placodes and buds in mouse
embryos. Relatively little is known of the signaling pathways that initiate
branching morphogenesis and the formation of the ducts from the embryonic
buds. Previous studies have shown that parathyroid hormone-related protein
(PTHrP; also known as parathyroid hormone-like peptide, Pthlh) is produced by
mammary epithelial cells and acts on surrounding mesenchymal cells to promote
their differentiation into a mammary-specific dense mesenchyme. As a result of
PTHrP signaling, the mammary mesenchyme supports mammary epithelial cell fate,
initiates ductal development and patterns the overlying nipple sheath. In this
report, we demonstrate that PTHrP acts, in part, by sensitizing mesenchymal
cells to BMP signaling. PTHrP upregulates BMP receptor 1A expression in the
mammary mesenchyme, enabling it to respond to BMP4, which is expressed within
mesenchymal cells underlying the ventral epidermis during mammary bud
formation. We demonstrate that BMP signaling is important for outgrowth of
normal mammary buds and that BMP4 can rescue outgrowth of PTHrP-/-
mammary buds. In addition, the combination of PTHrP and BMP signaling is
responsible for upregulating Msx2 gene expression within the mammary
mesenchyme, and disruption of the Msx2 gene rescues the induction of
hair follicles on the ventral surface of mice overexpressing PTHrP in
keratinocytes (K14-PTHrP). Our data suggest that PTHrP signaling sensitizes
the mammary mesenchyme to the actions of BMP4, triggering outgrowth of the
mammary buds and inducing MSX2 expression, which, in turn, leads to lateral
inhibition of hair follicle formation within the developing nipple sheath.

INTRODUCTION

In mice, mammary gland development begins with the formation of two
multilayered ridges of epidermal cells known as the mammary lines, which are
located between the fore and hind limb buds on either side of the ventral
surface of the embryo. The mammary lines are first discernable on embryonic
day 10 (E10) and, between E10.5 and E12.5, cells within these ridges are
thought to migrate to ten characteristic locations, where they invaginate into
the underlying mesenchyme to form the mammary buds. Between E15 and E16,
epithelial cells within the mammary buds begin to divide and give rise to the
mammary sprout, which grows out of the initial mesenchyme down through the
dermis and into the developing mammary fat pad. Once in this stromal
compartment, the nascent mammary duct begins to branch and by birth gives rise
to approximately 10-15 primary branches. This rudimentary duct system persists
until rising hormone levels at puberty trigger a second round of rapid
epithelial proliferation and ductal branching morphogenesis
(Hens and Wysolmerski, 2005;
Veltmaat et al., 2003;
Robinson et al., 1999).

The development of the embryonic mammary gland depends on a series of
reciprocal interactions between epithelial and mesenchymal cells, which guide
the formation of the placodes and buds, establish mammary cell fates and
initiate the three-dimensional morphogenesis necessary for the formation of
the primary ductwork (Hens and
Wysolmerski, 2005; Veltmaat et
al., 2003; Robinson et al.,
1999). Our understanding of the molecular events underpinning
early mammary development remains rudimentary, but recent work has begun to
characterize some of the mediators of these crucial epithelial-mesenchymal
interactions. Like in other organs, members of the FGF, hedgehog, WNT and EGF
growth factor signaling pathways play important roles in the patterning and
formation of the initial mammary placodes
(Chu et al., 2004;
Davenport et al., 2003;
Eblaghie et al., 2004;
Hatsell and Cowin, 2006;
Howard et al., 2005;
Mailleux et al., 2002;
Veltmaat et al., 2004;
Veltmaat et al., 2006). As
discussed below, parathyroid hormone related protein (PTHrP; also known as
parathyroid hormone-like peptide, Pthlh) and its receptor are important to the
formation of the mammary mesenchyme and outgrowth of the nascent mammary ducts
(Hens and Wysolmerski, 2005).
However, little else is known of the signaling pathways mediating the initial
wave of branching morphogenesis that gives rise to the primary duct
system.

PTHrP and the PTH1R are both required for normal mammary gland development.
Disruption of either gene in mice and loss of PTH1R function in humans results
in the complete absence of the mammary epithelium
(Wysolmerski et al., 1998;
Dunbar and Wysolmerski, 1999;
Foley et al., 2001). During
murine development, PTHrP is prominently expressed within mammary epithelial
cells, beginning on day E11 concurrent with the formation of the mammary
placodes. The PTH1R is expressed on immature mesenchymal cells located beneath
the entire epidermis. As the mammary bud is invaginating, PTHrP acts on its
receptor to induce the differentiation of the surrounding mesenchyme into the
specialized condensed mammary mesenchyme. Stimulation by PTHrP is required for
this mesenchyme to perform three vital functions: (1) to maintain the mammary
fate of the epithelial cells; (2) to trigger the overlying epidermis to form
the nipple sheath; and (3) to initiate ductal outgrowth and morphogenesis. In
the absence of PTHrP signaling, mammary epithelial cells differentiate into
skin cells, no nipple is formed and morphogenesis is interrupted
(Wysolmerski et al., 1998;
Dunbar et al., 1999;
Foley et al., 2001).
Conversely, overexpression of PTHrP in the basal keratinocytes of transgenic
mice using the keratin 14 promoter (K14-PTHrP mice) leads to the conversion of
the subepidermal mesenchyme from dermis into condensed mammary mesenchyme
(Foley et al., 2001). This, in
turn, suppresses hair follicle development and causes the epidermis to acquire
the characteristics of the nipple sheath. However, curiously, the epidermal
phenotype of the K14-PTHrP mice is restricted to the ventral surface of the
mouse between the borders of the original mammary lines, suggesting that this
area represents a specific zone of sensitivity to the effects of PTHrP
(Dunbar et al., 1999;
Foley et al., 2001).

MSX1 and MSX2 are BMP-responsive homeodomain-containing transcription
factors that have been shown to participate in the relay of signals between
epithelium and mesenchyme during development
(Phippard et al., 1996;
Satoh et al., 2004;
Satokata et al., 2000). Both
are particularly important to the normal development of epidermal appendages,
including the mammary gland. MSX1 and MSX2 are each expressed in the
epithelial cells of the forming mammary bud, and in mice with both genes
deleted, mammary development fails at the placode stage
(Satokata et al., 2000). MSX2,
but not MSX1, is also expressed within the dense mammary mesenchyme
surrounding the mammary epithelial cells, and after E14.5 its expression
becomes restricted to the mesenchymal compartment
(Phippard et al., 1996;
Satokata et al., 2000).
Similar to the phenotype of PTHrP-/- mice, mammary buds are
reported to form in MSX2-/- mice, but their development arrests at
E16.5 and no ductal outgrowth is formed
(Satokata et al., 2000).
Interestingly, activation of the PTH1R modulates Msx2 gene expression
in aortic adventitial cells and in osteoblasts
(Shao et al., 2003;
Shao et al., 2005;
Bidder et al., 1998;
Dodig et al., 1999;
Towler et al., 2006).

In this study, we examined potential interactions between PTHrP and BMP
signaling during early embryonic mammary gland development. We demonstrate
that PTHrP signaling is permissive for BMP signaling in the mammary
mesenchyme. This interaction, in turn, activates Msx2 gene expression
within the mesenchymal cells, which enables them to suppress hair follicle
formation in the overlying nipple skin. PTHrP and BMP signaling also cooperate
to enable the mesenchyme to initiate outgrowth of the mammary epithelial
buds.

MATERIALS AND METHODS

Animals

Animal experiments were approved by the Yale University IACUC.
MSX2+/- (tm1R1m) mice were obtained through the Mutant Mouse
Regional Resource Center (MMRRC) at the University of California, Davis and
MSX2-/- mice were identified as previously described
(Satokata et al., 2000).
Bmp4-lacZneo, K14-PTHrP, PTHrP-/- and
PTH1R-/- mice were identified as described previously
(Wysolmerski et al., 1998;
Foley et al., 2001;
Zhang et al., 2002).

Histology and immunohistochemistry

Histology and immunohistochemistry were performed using standard
techniques. Skin samples were fixed overnight in 4% paraformaldehyde or
Bouin's and were embedded in paraffin. Antigen retrieval was performed by
heating sections in 7 mM citrate under pressure, after which incubation with
primary antibody was carried out for 12 hours at 4°C. Antibodies to
filaggrin, K14 and tenascin C (Covance, Berkeley CA) were detected with the
Vector Elite ABC kits (Vector Laboratories, Burlingame. CA) and 3,3′
diaminobenzidine as a chromagen. β-Catenin antibodies (Transduction
Laboratories, Lexington KY) were detected with a goat anti-mouse
Alexa546-conjugated secondary antibody (Molecular Probes, Eugene, OR).

BMP4 is expressed in the ventral mesenchyme during embryonic mammary
gland development. (A) β-galactosidase staining of a
Bmp4-lacZneo embryo on E12.5. In these embryosβ
-galatosidase is expressed under the control of the BMP4 promoter. At
this stage of development, BMP4 is normally prominently expressed on the
ventral surface of the embryo. (B) Cross-section of an E13.5
Bmp4-lacZneo mammary bud. BMP4 is expressed in both the
mammary epithelium and the mammary mesenchyme, but more prominently within the
mesenchme (arrows). Note the absence of expression in the epidermis (E).

In situ hybridization

In situ hybridization was performed on 5 μm paraffin sections as
described previously (Dunbar et al.,
1998). The probe for MSX2 was a kind gift of Dr Richard Maas and
has been previously described (Satokata et
al., 2000). BMP receptor probes were a generous gift of Dr Vicki
Rosen (Boston, MA). Sense and antisense probes were generated from linearized
fragments using an in vitro transcription kit (Promega, Madison WI) in the
presence of [35S]UTP (1000 Ci/mmol, Amersham, Life Science,
Arlington Heights, IL).

RNA isolation and RT-PCR

Total RNA was isolated from cells or tissue using Trizol reagent (Gibco,
Gaithersville, MD) and samples were treated with DNase as described by the
manufacturer (GenHunter Corp., Nashville, TN). Quantitative RT-PCR (qRT-PCR)
was performed by standard methods using the OpticonII DNA engine (JM Research,
Waltham, MA). The mouse gene expression assay (Applied Biosystems, Foster
City, CA) was used for MSX2 (Mm00442992_m1). We generated the following primer
sets for SYBR-Green-based qRT-PCR: BMPr1a forward primer
5′-GGTATCTGGGTCAAAGCTGTTC-3′ and reverse primer
5′-CCTGCTGTCTCACTGGTGTAAG-3′, which spans nucleotides 87-244 of
the BMPr1a coding sequence (NM_009758). Probe-based qRT-PCR was performed with
Brilliant qRT-PCR master mix (Stratagene, La Jolla, CA) and SYBR-Green-based
qRT-PCR was performed with Brilliant SYBR-Green qRT-PCR master mix
(Stratagene). Samples were normalized for relative quantification of
expression by the 2-ΔΔCT method (Applied Biosystems
1997). Relative quantitation of gene expression: ABI Prism 7700 sequence
detection system, user bulletin 2, revision B. Samples were run in duplicate.
cDNA was prepared using the ABI PRISM as per the manufacturer's
instructions.

Embryo and mammary bud cultures

Freshly harvested E13 embryos were decapitated and placed on PET
track-etched membrane cell culture inserts containing 0.4 μm pores (Becton
Dickinson) in six-well plates. Embryos were cultured in F12/DMEM media
containing 10% FBS and antibiotics. Embryos were treated for 12-16 hours with
or without 10-7 M PTHrP (Sigma) after which ventral and/or dorsal
epidermis was dissected and used to make RNA.

In order to prepare bud cultures, individual mammary buds were
microdissected from E13 wild-type and PTHrP-/- embryos and placed
on Whatman 13 mm nuclepore Track-etched membranes (8 μm pore size; Thomas
Scientific, Swedesboro, NJ) on top of a tuft of ventral mesenchyme. All
dissections were performed in DMEM at 4°C. The filters were cultured on
EC587-40 mesh screen grills (Thomas Scientific, Swedesboro, NJ) in six-well
plates containing 10% FBS in DMEM/F12 media with antibiotics. Media was
changed every other day for the length of the experiment. Bud cultures were
fixed in acid alcohol and stained in carmine alum. Stained tissue was then
dehydrated and mounted in Permount (Fisher Scientific, NJ) for viewing.

RESULTS

PTHrP modulates BMP signaling in the embryonic mammary
mesenchyme

Overexpression of PTHrP in the basal keratinocytes of transgenic mice
(K14-PTHrP mice) causes dermal mesenchyme to become mammary-specific
mesenchyme, which, in turn, causes epidermis to become specialized nipple skin
(Foley et al., 2001). These
changes in cell fate are restricted to the ventral surface of the embryo,
despite the fact that both the K14-PTHrP transgene and the PTH/PTHrP receptor
are expressed within both the dorsal and ventral surfaces of the embryo.
Therefore, we hypothesized that the effects of PTHrP on mammary mesenchyme and
skin differentiation depend on the cooperation of some other, ventrally
restricted, signaling pathway. Recently, using a mouse in which the
lacZ gene was knocked into the Bmp4 gene locus, Zhang and
colleagues reported that BMP4 expression was strongly expressed within the
ventral surface of E13.5 embryos (Zhang et
al., 2002). Using the same model, we examined the expression of
BMP4 during mammary bud formation. As shown in
Fig. 1, BMP4 was expressed in
mesenchymal cells underlying the ventral epidermis between the four limb buds.
BMP4 was also expressed in epithelial cells of the mammary bud, but was not
expressed within keratinocytes. This pattern of expression was present between
E11.5 and E14.5, after which the lacZ expression became primarily
associated with developing hair follicles.

Because the pattern of BMP4 expression in the subepidermal mesenchyme was
essentially identical to the pattern of ectopic mammary mesenchyme in
K14-PTHrP embryos (Foley et al.,
2001), we next examined the possibility that PTHrP and BMP4
signaling interact during the formation of mammary buds. BMP signaling is
complex, but BMP4 is typically thought to induce the phosphorylation of
rSMADs, which includes SMADs 1, 5 and 8
(Massague and Chen, 2000).
Therefore, we examined the pattern of SMAD phosphorylation in wild-type
mammary buds and in genetic models of loss and gain of PTHrP function using an
antibody specific for the phosphorylated forms of these three SMADs. As
demonstrated in Fig. 2, we
performed immunohistochemistry on mammary buds from wild-type,
PTHrP-/- and PTH1R-/- mammary buds harvested at E15.5.
Wild-type buds were positive for nuclear phospho-SMAD 1, 5, 8 in both the
epithelial and mesenchymal compartments
(Fig. 2B). Furthermore, only
the mammary mesenchyme and not the general dermal mesenchyme stained. By
contrast, in PTHrP-/- and PTH1R-/- buds, phospho-SMAD 1,
5, 8 staining was significantly reduced in the mammary mesenchyme compared
with the wild-type buds (Fig.
2A,C). We also compared phospho-SMAD 1, 5, 8 staining in the
ventral skin of K14-PTHrP mice to that in wild-type ventral skin. As shown in
Fig. 2D, there was little
phospho-SMAD staining in the dermis of wild-type mice at E18.5, but there was
prominent staining in the ectopic mammary mesenchyme beneath the ventral skin
of K14-PTHrP littermates (Fig.
2E). Dorsal skin from wild-type and K14-PTHrP mice showed
phospho-SMAD staining associated with developing hair follicles and their
associated mesenchyme, but there was no significant staining in the
interfollicular dermis of either genotype (data not shown). These results
demonstrate that alterations of PTHrP signaling in embryonic mammary buds and
skin lead to changes in SMAD phosphorylation within mesenchymal cells, but
only within the ventral zone of BMP4 expression in vivo, suggesting that PTHrP
signaling may interact with BMP signaling in these cells.

PTHrP augments BMP signaling in vivo and in vitro. (A-E)
Immunohistochemistry for phospho-SMAD 1, 5, 8 in sections through a
PTHrP-/- mammary bud at E15.5 (A), a wild-type bud at E15.5 (B), a
PTH1R-/- bud at E15.5 (C), wild-type ventral skin at E18.5 (D) and
K14-PTHrP ventral skin at E18.5 (E). Note the nuclear staining for
phospho-SMADs in the wild-type mammary mesenchyme (arrows in B) and its
absence in the mammary mesenchyme of PTHrP-/- and
PTH1R-/- buds (A,C). Also note the induction of nuclear
phospho-SMAD staining in the mesenchyme of the ventral skin in K14-PTHrP
embryos (arrows in E). (F) Western blot for phospho-SMAD 1, 5, 8 of
whole cell lysates from C3H10T1/2 cells treated overnight with graded BMP4
concentrations (0, 10, 50, 100 ng/ml BMP4) with or without 10-7 M
PTHrP. The addition of PTHrP augments phospho-SMAD expression (representative
blot of five experiments).

PTHrP sensitizes mesenchymal cell lines to the effects of BMP

In order to see if there was a direct interaction between PTHrP and BMP
signaling, we treated two well-characterized mesenchymal cell lines with both
PTHrP and BMP2 or 4. We first confirmed that C3H10T1/2 and C2C12 cells
expressed the PTH1R (data not shown). Next we assayed SMAD phosphorylation in
both cell lines treated for 18-20 hours with varying doses (0-100 ng/ml) of
BMP4 or BMP2 in the presence or absence of 10-7 M PTHrP 1-34.
Fig. 2F shows the results for
C3H10T1/2 cells treated with BMP4. Similar results were obtained with BMP2 and
BMP4 in C2C12 cells (data not shown). In the absence of PTHrP, increasing
doses of BMP resulted in a progressive increase in SMAD phosphorylation. The
addition of PTHrP sensitized these cells to BMP treatment such that the levels
of phospho-SMAD 1, 5, 8 were increased at each BMP concentration used
(Fig. 2F). These data suggest
that PTHrP signaling interacts directly with BMP signaling within mesenchymal
cells.

PTHrP augments BMP signaling through regulation of BMPR1A
expression

We next attempted to define a mechanism by which PTHrP might sensitize the
mammary mesenchyme to the actions of BMP. A previous report had suggested that
PTHrP increased the expression of the BMPR1A in C3H10T1/2 cells as assessed by
Northern blot (Chan et al.,
2003). We confirmed this observation by performing qRT-PCR on RNA
prepared from C3H10T1/2 cells treated with 10-7 M PTHrP for 18-20
hours. As seen in Fig. 3A, this
resulted in an approximate doubling of BMPr1a mRNA levels. We next asked if
PTHrP was able to increase the expression of BMPr1a mRNA in mammary mesenchyme
in vivo. First, we performed in situ hybridization to examine BMPr1a
expression in embryonic mammary buds. Fig.
3E,F demonstrates the results for wild-type buds at E15.5. As one
can see, BMPr1a mRNA was expressed at low levels throughout the entire
subepidermal mesenchyme, including the dense mammary mesenchyme. It was
difficult to determine if there was specific expression within the epidermis,
but the gene was not expressed within mammary epithelial cells at this stage
of development. We did not detect clear differences in expression of BMPr1a
within the mammary mesenchyme of PTHrP-/- buds or within the
ectopic mammary mesenchyme beneath the ventral epidermis of K14-PTHrP mice by
in situ hybridization (data not shown). Because this receptor appeared to be
expressed at low levels in the mesenchyme and because in situ hybridization is
not a sensitive quantitative technique, we also addressed this question by
performing qRT-PCR on samples of skin and mammary buds microdissected from
pharmacologically and genetically manipulated embryos. Wild-type E13.5 embryos
were cultured for 18-20 hours in the presence or absence of 10-7 M
PTHrP. The ventral epidermis and its associated mesenchyme were then removed
and assayed for BMPr1a mRNA expression by qRT-PCR. As seen in
Fig. 3B, PTHrP treatment
resulted in an approximate doubling of BMPr1a gene expression in the
ventral epidermis, a result similar to its effects on BMPr1a gene
expression in C3H10T1/2 cells. BMPr1a mRNA expression also was
increased in ventral skin isolated from K14-PTHrP embryos at day E18
(Fig. 3C). Finally, we examined
the relative levels of BMPr1a mRNA in freshly isolated mammary buds
from E15.5 wild-type and PTHrP-/- embryos. As seen in
Fig. 3D, BMPr1a mRNA
levels were reduced by 75% in the PTHrP-/- buds compared with
wild-type buds. As the Pth1r and BMPr1a genes are expressed
on mesenchymal cells in embryonic skin and mammary buds, these data suggest
that PTHrP secreted by the embryonic mammary buds regulates expression of the
BMPr1a gene in mammary mesenchyme.

PTHrP induces BMPr1a mRNA expression in vitro and in vivo.
(A) BMPr1a mRNA detected by qRT-PCR in C3H10T1/2 cells treated with or
without 10-7 M PTHrP overnight (n=3 experiments).
(B) qRT-PCR for BMPr1a mRNA in ventral skin from cultured wild-type
E13.5 embryos treated with or without 10-7 M PTHrP overnight (three
experiments with two to three embryos per treatment). Note that PTHrP
increases BMPr1a expression. (C) qRT-PCR for BMPr1a mRNA in the ventral
skin from E18.5 K14-PTHrP transgenic and wild-type control mice. BMPr1a
expression is higher in K14-PTHrP epidermis (n=4 experiments).
(D) qRT-PCR for BMPr1a mRNA in mammary buds microdissected from either
wild-type or PTHrP-/- embryos on E15.5. Expression is reduced in
the PTHrP knockout buds compared with wild-type controls. RNA was from pooled
buds (50-100 buds per sample). (E,F) Dark-field (E) and
corresponding light-field (F) images of in situ hybridization for BMPr1a
expression in an E15.5 wild-type mammary bud. Arrowhead points to the
epithelial bud. The BMPr1a gene is expressed at a low level throughout the
mesenchyme, but appears not to be expressed within the mammary epithelium.

BMP4 rescues outgrowth of PTHrP-/- mammary buds in organ
culture. (A-D) Representative examples of bud outgrowths after 7
days in organ culture. (A) Wild-type mammary bud. (B) PTHrP-/- bud.
(C) PTHrP-/- bud treated with 10-7 M PTHrP. (D)
PTHrP-/- bud treated with 10 ng/ml BMP4. (E) Quantification
of the frequency of bud outgrowth under different conditions as noted on the
x-axis. Each experiment was performed on 15 buds over three separate
experiments, except for the wild-type buds, which represent a total of 40 buds
over eight separate experiments. *, P<0.05 for WT
versus WT+noggin; #, P<0.05 for WT versus KO; triangle,
P<0.05 for KO versus KO+PTHrP and KO versus KO+BMP4.

BMP4 can rescue outgrowth of mammary buds in PTHrP-/-
embryos

Our next question concerned the physiological relevance of the ability of
PTHrP to augment BMP signaling in the embryonic mammary bud. We reasoned that
if BMP4 acted in a pathway downstream of PTHrP in the mammary bud, then
supplying it to PTHrP-/- buds might rescue their developmental
arrest. In order to test this possibility, we developed a mammary bud culture
system, which allowed us to recapitulate the PTHrP-/- bud
phenotype. As adapted from Robinson et al.
(Robinson et al., 2000),
mammary buds were isolated from wild-type and PTHrP-/- embryos and
were cultured with extra tufts of ventral mesenchyme on nucleopore filters.
After 7 days in the presence or absence of 10-7 M PTHrP, or 5 ng/ml
or 10 ng/ml BMP4, cultures were scored for bud outgrowth. A bud was considered
to have grown out normally if the length of the initial sprout was more then
the length of the bud itself and if at least one branch point had formed.
Fig. 4A-D demonstrate the
typical appearance of the cultures, and
Fig. 4E shows the
quantification of outgrowth from multiple bud cultures. As can be seen, after
7 days in culture, wild-type buds produced an elongated sprout with several
primary branches in approximately 70% of the cultures
(Fig. 4A,E). The degree of
branching of the outgrowths was variable but reminiscent of the normal
embryonic duct system at E18.5 (Hens and
Wysolmerski, 2005). By contrast, as depicted in
Fig. 4B, the majority of
PTHrP-/- buds failed to sprout, with only 10% of the buds showing
outgrowth (Fig. 4E). Growth of
PTHrP-/- buds was restored by the addition of PTHrP to the media
(45% of cultures sprouted, Fig.
4C,E). In addition, treatment of PTHrP-/- bud cultures
with BMP4 also rescued bud outgrowth so that 56% of buds met our sprouting
criteria (Fig. 4D,E). The
addition of equivalent concentrations of PTHrP or BMP4 to wild-type buds had
no effect on bud outgrowths (data not shown). Finally, we examined the effects
of inhibiting BMP signaling on the growth of wild-type buds by using the
soluble BMP inhibitor, noggin (Rosen,
2006; Botchkarev et al.,
1999). As shown in Fig.
4E, the addition of noggin, a secreted BMP inhibitor, to wild-type
buds reduced the likelihood of outgrowth by half so that only 33% of
noggin-treated wild-type buds sprouted compared with the 68% of control buds
that sprouted. These data demonstrate that both PTHrP and BMP signaling are
important to the initiation of normal bud outgrowth. Furthermore, the ability
of BMP4 to complement the loss of PTHrP in mammary bud cultures strongly
suggests that BMP4 signaling acts downstream of PTHrP to initiate ductal
branching morphogenesis from embryonic mammary buds.

PTHrP regulates MSX2 expression in mammary mesenchyme

MSX2 is a homeodomain-containing transcription factor known to be involved
in epithelial-mesenchymal interactions during development. It is thought to
have important functions during the formation of epidermal appendages, as
disruption of the Msx2 gene results in abnormal tooth, hair follicle
and mammary gland development (Satokata et
al., 2000). In fact, mammary development in MSX2-/-
mice has been reported to arrest after the formation of an apparently normal
bud, a phenotype remarkably similar to that seen in PTHrP-/-
embryos (Satokata et al.,
2000). Furthermore, in vascular cells, MSX2 expression is
regulated by both BMPs and parathyroid hormone
(Shao et al., 2003;
Shao et al., 2005). For these
reasons, we hypothesized that MSX2 might mediate the effects of combined PTHrP
and BMP signaling in the mammary bud.

In order to explore this hypothesis, we first examined Msx2 expression in
wild-type, PTHrP-/- and PTH1R-/- mammary buds in vivo by
in situ hybridization. As previously described, we found the Msx2
gene to be expressed within the mammary mesenchyme in wild-type buds at E15.5
(Fig. 5B,E)
(Satokata et al., 2000;
Phippard et al., 1996).
Interruption of PTHrP signaling through disruption of either the
Pthrp or Pth1r genes resulted in a significantly reduced
level of Msx2 mRNA in the mammary mesenchyme
(Fig. 5A,D,C,F). Furthermore,
Msx2 gene expression was prominently and ectopically induced in the
ventral dermis by expression of PTHrP in the basal keratinocytes of K14-PTHrP
transgenic mice (compare Fig. 5G,H with
5I,J). Thus, in vivo, MSX2 expression correlates with alterations
in PTHrP signaling as well as the parallel alterations of BMP signaling
demonstrated in Fig. 2.

We next examined changes in Msx2 mRNA levels in response to PTHrP
and BMP signaling in C3H10T1/2 cells in order to test directly if PTHrP and
BMP signaling interact to regulate Msx2 expression. As shown in
Fig. 6, treatment of the cells
with either PTHrP or BMP4 alone modestly stimulated Msx2 mRNA
expression. However, the combination of PTHrP and BMP4 augmented Msx2
expression to a much greater extent. These data again demonstrate that PTHrP
and BMP signaling interact and identify the Msx2 gene as a target of
the cooperative interaction between the two.

PTHrP and BMP4 interact to regulate Msx2 gene expression in
C3H10T1/2 cell. qRT-PCR for Msx2 mRNA in C3H10T1/2 cells treated
for 7 days with 10-7 M PTHrP, 50 ng/ml BMP4, or both together.
Shown is the average level of expression represented as the fold induction
over no treatment (NT). Each bar represents the average±s.e.m. of three
individual experiments.

MSX2 mediates the effects of PTHrP on hair follicle development in
vivo

In order to determine if MSX2 mediated the effects of the combined actions
of PTHrP and BMP on the mammary mesenchyme, we bred the K14-PTHrP transgene on
to an MSX2-/- background. Our reasoning was that if MSX2 were
important to the functions of PTHrP, then removing MSX2 should prevent the
effects of PTHrP overexpression. As shown in
Fig. 7B, overexpression of
PTHrP in basal keratinocytes converts the epidermis into nipple skin and the
ventral dermis into condensed mammary mesenchyme
(Foley et al., 2001). As a
result, the ventral epidermis of K14-PTHrP mice is thickened, lacks hair
follicles and displays characteristic alterations in keratin and filaggrin
expression. In addition, the dermis in these mice is hypercellular and
expresses markers usually restricted to the mammary mesenchyme
(Foley et al., 2001). As is
evident from Fig. 7, disruption
of the Msx2 gene mitigated the K14-PTHrP phenotype but did not
completely reverse it. The most striking finding was the recovery of hair
follicle development in the K14-PTHrP/MSX2-/- embryos
(Fig. 7C). Although deletion of
MSX2 has been reported to cause abnormal hair cycling, the initial development
of follicles in the embryo is normal
(Satokata et al., 2000;
Ma et al., 2003). As one can
see, removing MSX2 from the K14-PTHrP epidermis led to the recovery of hair
follicle induction in the ventral skin. In addition, there was some resolution
of the epidermal thickening and the dermis was less compact in
K14-PTHrP/MSX2-/- embryos compared with K14-PTHrP controls.
However, there was no change in epidermal expression of keratin 14 or
filaggrin in the ventral skin of K14-PTHrP/MSX2-/- embryos compared
with the changes in the expression of these markers previously documented in
the ventral mesenchyme of K14-PTHrP embryos (data not shown). Furthermore,
K14-PTHrP/MSX2-/- embryos continued to express ectopic markers of
the mammary mesenchyme such as LEF1, β-catenin, androgen receptor and
tenascin C in the ventral dermis, despite the clear morphological changes
noted above (data not shown). These data identify MSX2 as a crucial
mesenchymal factor that allows PTHrP and BMP4 to suppress hair follicle
induction in the vicinity of the developing mammary bud and nipple.

DISCUSSION

Our data document interactions between PTHrP and BMP signaling during
embryonic mammary gland development. Previous studies had shown that PTHrP
regulates a series of cell fate decisions necessary for the differentiation of
the mammary mesenchyme, the formation of the nipple and the initiation of
branching morphogenesis from the mammary bud
(Wysolmerski et al., 1998;
Dunbar et al., 1998;
Foley et al., 2001). The
current study suggests that PTHrP acts, at least in part, by modulating BMP
signaling in mammary mesenchyme cells. We show that during the formation of
the mammary buds, BMP4 is expressed by mesenchymal cells on the ventral
surface of the embryo. PTHrP signaling sensitizes mesenchymal cells to BMP in
vitro and in vivo, by increasing the expression of the BMP1A receptor. The
interaction between PTHrP and BMP4 is important for mammary bud sprouting and
BMP4 treatment can rescue outgrowth of PTHrP-/- mammary buds in
organ culture. Finally, we demonstrate that the combination of PTHrP and BMP
signaling increases the expression of the homeobox gene, MSX2, in the mammary
mesenchyme, which, in turn, allows PTHrP to suppress hair follicle formation
in the vicinity of the developing mammary bud and nipple.

Our data showing that PTHrP signaling upregulates the expression of the
BMPR1A in the mammary mesenchyme are similar to those of Chan and colleagues
(Chan et al., 2003), who
demonstrated an increase in the expression of this receptor in response to
PTHrP in the pluripotent mesenchymal cell line, C3H10T1/2. Using qRT-PCR, we
showed an approximate doubling of Bmpr1a expression in these same
cells when they were treated with PTHrP. In addition, we saw a doubling of
Bmpr1a mRNA expression in the skin of embryos treated with PTHrP ex
vivo as well as in the epidermis of K14-PTHrP embryos overexpressing PTHrP in
basal keratinocytes. We also noted a 75% reduction in the level of
Bmpr1a mRNA in mammary buds microdissected from PTHrP-/-
embryos compared with buds derived from wild-type littermates, demonstrating
that PTHrP expression by mammary epithelial cells is important to the native
level of BMPR1A expression surrounding the buds. Although we could not detect
a difference in Bmpr1a gene expression by in situ hybridization,
these experiments did demonstrate that the receptor is expressed in
mesenchymal cells and not in mammary epithelial cells during early bud
development, suggesting that the alterations in expression noted above are
likely to be in the mammary mesenchyme surrounding the developing bud and in
the ectopic mammary mesenchyme that forms under the epidermis in K14-PTHrP
embryos. This conclusion is also consistent with the localization of PTH1R
expression in mesenchymal cells at this stage of development
(Dunbar et al., 1998).
Finally, results from a model of transgenic overexpression of PTHrP in
developing lung also demonstrate upregulation of Bmpr1a gene
expression in response to PTHrP (W. Philbrick, personal communication). Thus,
PTHrP may regulate BMP receptor expression in several organs and modulation of
BMP signaling may be a more general feature of the actions of PTHrP during
development.

PTHrP and BMP signaling interact to initiate mammary bud outgrowth and
nipple formation. PTHrP is secreted from mammary epithelial cells and
increases BMPR1A expression in the mammary mesenchyme. This increases the
sensitivity of these cells to BMPs and allows them to respond to BMP4 in a
paracrine and/or autocrine fashion. BMP4 signaling in the mesenchyme, in turn,
triggers epithelial outgrowth and augments MSX2 expression, which causes the
mammary mesenchyme to inhibit hair follicle formation within the nipple
sheath.

Overexpression of PTHrP in basal keratinocytes in K14-PTHrP mice leads to a
transformation of dermal mesenchyme into mammary mesenchyme, which, in turn,
suppresses the formation of hair follicles and alters the differentiation of
keratinocytes (Foley et al.,
2001). Interestingly, these changes occur only on the ventral
surface of the embryo, despite the fact that the K14-PTHrP transgene is
expressed in both ventral and dorsal keratinocytes and that the PTH1R is
expressed on mesenchymal cells in the dermis on both surfaces. In addition, it
has been shown that dermal mesenchyme harvested from the ventral surface of
the embryo responds to the mammary epithelial bud by upregulating expression
of the androgen receptor (a marker of the mammary mesenchyme), whereas
mesenchyme from the dorsal skin does not
(Heuberger et al., 1982)
(G.W.R. and K. Kratochwil, unpublished). Our findings offer a potential
explanation for these observations. We propose that PTHrP acts to promote
mammary mesenchyme differentiation and bud outgrowth by modulating mesenchymal
cell responsiveness to BMPs. Therefore, the presence of a specific BMP is
needed for the full expression of the effects of PTHrP. In essence, the
pattern of BMP4 expression creates a ventrally restricted zone of
responsiveness to the effects of PTHrP. In the case of normal mammary
development, the expression of BMP4 in the ventral mesenchyme coincides with
the formation of the mammary buds, which express PTHrP and allows both
signaling pathways to interact to form the mammary-specific mesenchyme. In the
setting of the K14-PTHrP transgene, ectopic formation of mammary mesenchyme is
restricted to the ventral area of BMP 4 expression.

Deletion of the PTHrP gene in mice and humans leads to a failure of the
mammary bud to initiate branching morphogenesis
(Wysolmerski et al., 1998;
Wysolmerski et al., 2001).
Culture of PTHrP-/- mammary buds ex vivo recapitulates the failure
of bud outgrowth in vivo. While 70% of wild-type mammary buds initiated
branching growth in culture, only 10% of PTHrP-/- buds did so, a
defect rescued by the addition of PTHrP to the culture media. Significantly,
the addition of BMP4 to the bud cultures was able to complement the loss of
PTHrP and also rescue outgrowth of PTHrP-/- buds. Furthermore,
noggin treatment was able to inhibit the growth of wild-type buds. These
experiments suggest that BMP4 acts downstream of PTHrP to initiate outgrowth
of the mammary buds. BMP4 has been shown to regulate branching morphogenesis
in several other organs including the lung, submandibular gland, prostate,
kidney and ureter (Eblaghie et al.,
2006; Shao et al.,
2005; Martinez et al.,
2002; Miyazaki et al.,
2000; Bellusci et al.,
1996; Bragg et al.,
2001; Weaver et al.,
2000; Lamm et al.,
2001; Dean et al.,
2004; Shi et al.,
2001). Its role has been best studied in the lung, where it
appears either to stimulate or inhibit branching, depending on the
experimental context (Eblaghie et al.,
2006; Shao et al.,
2005; Bellusci et al.,
1996; Bragg et al.,
2001; Weaver et al.,
2000; Shi et al.,
2001). Some of these conflicting effects may be related to the
level of BMP signaling or to differing effects on epithelial cells versus
mesenchymal cells. In the mammary bud, our results suggest that PTHrP-mediated
upregulation of BMPR1A expression allows for spatially restricted, autocrine
or paracrine BMP signaling within the mesenchyme. We believe that this BMP
signal, in turn, enables mammary mesenchyme cells to trigger and/or support
outgrowth of the bud epithelium.

In addition to promoting bud outgrowth, the mammary-specific mesenchyme
instructs the overlying epidermis to form the nipple sheath, an activity that
is also dependent on PTHrP signaling. A prominent feature of the nipple sheath
is its lack of hair, which is probably the result of lateral inhibition of
hair follicle formation by PTHrP secreted by the epithelial bud. In the
absence of PTHrP or the PTH1R, hair follicles develop too close to the mammary
bud, and in the presence of PTHrP misexpression by basal keratinocytes, hair
follicle development is suppressed throughout the entire ventral epidermis
(Foley et al., 2001;
Wysolmerski et al., 1994). We
now find that the ability of PTHrP to suppress hair follicle development
depends on the actions of the homeobox transcription factor MSX2. Confirming
previous reports, using in situ hybridization we found that MSX2 was expressed
within the mammary mesenchyme (Phippard et
al., 1996; Satokata et al.,
2000). Furthermore, mesenchymal expression of MSX2 requires PTHrP
signaling, because MSX2 levels were reduced around PTHrP-/- buds
and MSX2 expression was induced within the ectopic mammary mesenchyme formed
beneath the ventral epidermis in K14-PTHrP transgenic mice. The ventral
restriction of MSX2 induction in these mice suggests that this transcription
factor is a specific target of the interaction between PTHrP and BMP signaling
discussed previously. This is not surprising, given the fact that MSX2 is
known to be regulated by BMPs in several sites during development
(Kratochwil et al., 1996;
Andl et al., 2004;
Towler et al., 2006;
Hussein et al., 2003). It is
also consistent with our data in vitro showing that while PTHrP or BMP4 alone
have only a modest effect on MSX2 expression in C3H10T1/2 cells, the
combination has a robust inductive effect on its expression. Given these
results, it will be interesting to determine whether PTHrP might potentiate
the effects of BMPs on MSX2 expression in other developing organs as well.

BMPs are known to suppress hair follicle formation, and antagonism of BMP
signaling in the mesenchyme by secreted BMP inhibitors is thought to be
important for the induction of at least some classes of hair follicles
(Kobielak et al., 2003;
Botchkarev et al., 2002;
Andl et al., 2004;
Botchkarev et al., 1999).
Furthermore, BMP signaling is thought to be important for lateral inhibition
and spacing of hair follicles and feathers
(Jung et al., 1998;
Noramly et al., 1999;
Mou et al., 2006). Therefore,
the sensitization of mesenchymal cells to BMP4 signaling by PTHrP might be
expected to suppress hair follicle formation, as happens on the ventral
surface of K14-PTHrP mice and around the mammary buds and nipple. Because
crossing the K14-PTHrP transgene onto an MSX2-/- background led to
the recovery of hair follicle formation in the ventral surface of
K14-PTHrP/MSX2-/- mice, it would also appear that PTHrP and BMP4
interact to suppress hair development by inducing Msx2 in the mammary
mesenchyme. Like mammary glands, hair follicle induction requires reciprocal
interactions between epithelial and mesenchymal cells. Our findings suggest
that MSX2 is able to interfere with the ability of the mesenchyme to induce
the formation of hair placodes. While MSX2 has previously been reported to be
necessary for the outgrowth of the mammary bud in mice, in our hands
MSX2-/- mice are able to form normal mammary ducts
(Satokata et al., 2000).
Furthermore, the expression of several markers of mammary mesenchyme
differentiation was also normal in MSX2-/- mammary buds (J.R.H. and
J.W., unpublished). Thus, MSX2 appears to be relatively specific for mediating
the hair-suppressing effects of PTHrP.

In closing, the experiments detailed in this report demonstrate an
important interaction between PTHrP and BMP4 during the development of the
embryonic mammary bud. As illustrated in
Fig. 8, PTHrP is secreted by
the mammary epithelial cells in the bud and interacts with its receptor on
surrounding mesenchymal cells. In response, these cells upregulate BMPR1A
expression and become able to respond in an autocrine and/or paracrine fashion
to mesenchymal BMP4 found within the ventral epidermis. As a result of this
cooperation between PTHrP and BMP signaling, the mammary-specific mesenchyme
forms and exerts its actions on promoting outgrowth of the primary mammary
ducts and instructing nipple sheath development. The suppression of hair
follicle formation in the epidermis immediately surrounding the nipple is
mediated by the induction of MSX2 expression in mesenchymal cells. It is
likely that other growth factors and/or transcription factors will also be
regulated by these pathways in the mammary mesenchyme, and it will be
particularly interesting to define which factor(s) allows the mesenchyme to
initiate outgrowth of the epithelial bud.

Acknowledgments

The authors would like to thank Drs Sarah Millar, William Philbrick and
Arthur Broadus for helpful discussions. This work was supported by grants
RO1DK55501 and RO1DK69542 from the NIH. Support was also provided by core
facilities funded by the Yale Diabetes and Endocrine Research Center
(P30DK45735) and the Yale Core Center for Musculoskeletal Disorders
(P30AR46032). J.R.H. received support from T32DK07058 to the Yale Section of
Endocrinology and Metabolism. G.W.R. is supported by the intramural program at
NIDDK of the NIH.